In medicine, the need for transfusional fluids is continually increasing. The use of stroma-free hemoglobin (Hb) solutions as a red cell replacement has been considered. Solutions of stroma-free Hb contain tetrameric (MW 64 kDa) and dimeric (MW 32 kDa) Hb molecules at equilibrium. Due to the rapid filtration of the dimers through the kidneys, the retention time of infused Hb solutions is short. In addition, Hb molecules extravasate through the endothelium, scavenging the NO from the interstitial fluid. The latter is believed to be the main reason for the increase in mean arterial pressure observed upon administration of a stroma-free Hb solution.
Chemical modifications have been used to transform mammalian Hbs into efficient blood substitutes, but the introduction of such chemical modifications can result in toxicity and/or an immunogenic response. For example, much work has been devoted to the preparation of Hb solutions with oxygen affinities similar to that of whole blood. Recent studies, however, indicate that Hb with high oxygen affinity can efficiently deliver O2 to the tissues. Thus, the question of optimal oxygen affinity for blood substitutes has not been resolved.
Adult Hb is a tetrameric protein comprised of two structurally similar subunits, α and β, assembled through two different interfaces. Each subunit contains eight α-helices (labeled A-H) that form a pocket containing the heme. The heme pocket of each subunit is lined by hydrophobic residues, except for the proximal (F8) and distal (E7) histidines, which are critical to the functional properties of Hb. The functional properties of the heme pocket are also greatly sensitive to the polar character of the amino acid side chains lining the pocket.
As previously mentioned, problems related to the use of Hb solutions for transfusion include the rapid loss of Hb through the kidneys and vasoconstriction with an increase in arterial blood pressure, which is thought to be due to scavenging of NO released from the endothelium. Moreover, at the physiological colloid-osmotic pressure of human plasma, only a limited amount of Hb may safely be infused, and thus the oxygen-carrying capacity of blood cannot be fully restored. In an effort to prevent these effects, tetramers of Hb molecules that resist dissociation into dimers in serum have been produced by using bifunctional reagents to effect intramolecular crosslinking. In the absence of further intermolecular crosslinking, stabilized tetrameric Hbs still extravasated across the endothelium and failed clinical tests. See, e.g., Saxena et al., 1999, Stroke. 30: 993-996 and Sloan et al., 1999, JAMA 282: 1857-1864.
Myoglobin (Mb) is a 17.5 kilodalton monomeric heme protein found mainly in muscle tissue, where it serves as an intracellular storage site for oxygen. During periods of oxygen deprivation, oxymyoglobin releases its bound oxygen which is then used for metabolic purposes.
The tertiary structure of Mb is that of a typical water soluble globular protein, and contains approximately 75% α-helical secondary structure. A Mb polypeptide is comprised of eight separate right handed α-helices, designated A through H, that are connected by short non-helical regions. Amino acid R-groups oriented towards the interior of the molecule are predominantly hydrophobic in character, and those on the surface of the molecule are generally hydrophilic, thus making the molecule relatively water soluble. Each Mb molecule contains one heme prosthetic group inserted into a hydrophobic cleft in the protein. To date, an adequate blood substitute which utilizes Mb is not available.
The curve of oxygen binding to Hb is sigmoidal, which is typical of allosteric proteins in which the substrate, in this case oxygen, is a positive homotropic effector. When oxygen binds to the first subunit of deoxyhemoglobin, the first oxygen molecule increases the affinity of the remaining subunits for additional oxygen molecules. As additional oxygen is bound to the other Hb subunits, oxygen binding is incrementally strengthened, so that Hb is fully oxygen-saturated at the oxygen tension of lung alveoli. Likewise, oxygen is incrementally unloaded and the affinity of Hb for oxygen is reduced as oxyhemoglobin circulates to deoxygenated tissue.
In contrast, the oxygen binding curve for Mb is hyperbolic in character, indicating the absence of allosteric interactions in this process. Mb therefore has a higher oxygen affinity and lower cooperativity than Hb. A large array of Mb heme pocket mutants have been constructed and investigated (Springer et al., Chem Rev 1994; 94: 699-714), and information is thus available on the molecular control of the conformational and functional properties of Mb. While these studies are certainly useful in the design of Hb-based oxygen carriers (Dou et al., Biophys Chem 2002; 98: 127-148), heme pocket differences exist between Mb and Hb. The effect of a particular mutation in Mb is therefore not necessarily predictive of the effect the analogous mutation would have in Hb.